This is the second report for this project. The project consists of the creation of 2 important tools for measuring T-cell receptor diversity. The first, is the use of high-throughput sequencing to measure the diversity of T-cell receptor rearrangements. The strategies and protocol development are ongoing and being done in collaboration with Danny Douek's lab in the NIH VRC. THe sequencing method, and techniques for analysis are nearly completed and patient samples will soon be analyzed using this method. The second tool is a functional measurement of diversity by stimulating patient T-cell samples with peptide libraries. This project is now very mature, as we are able to measure T-cell responses to random peptides in both the naive and memory compartments. We are now validating this method in patients with restricted repertoires. These two tools will be used to better characterize the TCR diversity of two important patient groups. The first is patients whose infectious and immune dysfunctional history is consistant with a T-cell defect, but when lymphocyte subsets are measured, no T-cell deficiency is noted. We hope to define holes in the TCR repertoires of these patients which could explain their infections. The second is in severely atopic patients, whose repertoires may also have similar "holes" but for whom the remaining reactive T-cells therefore develop an abberantly atopic, as opposed to absent response, to a given antigen. These patient cohorts are being assembled now, and as one might expect there are patients in these cohorts with both atopy and infecious predilection. We also hope to apply T-cell receptor repertoire studies to allergen-reactive T-cells, and have already begun to obtain data regarding the TCR sequences of Th1 versus Th2 allergen reactive T-cells. Furthermore, priming of polyclonal, naive human T-cells in vitro with peptide has never been accomplished. We are now able to do so, which will allow for the evaluation of early T-cell signaling and T helper cell differentiation in a fashion never previously done.